Recipient Organization
OHIO STATE UNIVERSITY - VET MED
1900 COFFEY ROAD, 127L VMAB
COLUMBUS,OH 43210
Performing Department
Preventive Medicine
Non Technical Summary
Porcine deltacoronavirus (PDCoV) is a virus that causes diarrhea in nursing piglets. We have evidence that PDCoV has the ability to jump from pigs to poultry and vice versa. Currently we don't know if PDCoV is already infecting poultry. The first steps in determining whether PDCoV is a threat to the poultry industry is to determine if poultry are already being infected. To assess whether infection is occurring we look at the individual's immune response to determine whether they have antibodies against PDCoV. We are proposing to develop a test to look at the blood of poultry to see whether they have been exposed to the virus. Once we have a test to specifically find PDCoV infected birds we can look at many serum samples to determine how frequently poultry are infected, determining whether they are at risk of contracting the disease and spreading it to other animals or humans.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
As a newly emerged zoonotic pathogen, little is known about the epidemiological aspects of PDCoV infection. We don't fully understand which species besides the pig are naturally infected by PDCoV. There are two main possibilities with commercial poultry and their link to PDCoV in swine. First, the existence of PDCoV in poultry flocks may be widespread causing little noticeable morbidity, potentially serving as a hidden reservoir fortransmission of PDCoV to other species. Second, poultry may not have been exposed to PDCoV to significant extent yet. In this scenario a situation exists whereby PDCoV may be poised to jump into a poultry flocks, potentially causing commercial losses due to decreased weight gain in birds, and through other potential morbidities. To test whether PDCoV is currently circulating in the commercial poultry industry we will screen commercial flocks for seroprevalence to PDCoV using a luciferase immunoprecipitation assay system (LIPS) followed by testing for PDCoV RNA in positive flocks though the following 2 Specific Aims:Aim 1: Develop a PDCoV specific luciferase immunoprecipitation assay system (LIPS) allowing assessment of seroprevalence of PDCoV antigens.Aim 2: Evaluate the exposure levels of commercial poultry flocks to PDCoV infection utilizing LIPS.
Project Methods
Aim 1: Develop a POCoV specific luciferase immunoprecipitation assay system (LIPS) allowing assessment of seroprevalence of PDCoV antigens.Luciferase immunoprecipitation system (LIPS). The LIPS assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies (5). UPS involves quantitating serum antibodies by measuring luminescence emitted by a reporter enzyme, nano luciferase (Niue), fused to an antigen of interest. The Niue-antigen fusion protein is recognized by antigen-specific antibodies, and antigenantibody complexes are captured by protein A/G beads that recognize the Fe region and capture the lgG or chicken lgY precipitating resin (Genscript) to capture chicken lgY. To Jssess the seroprevalence of poultry to PDCoV we will develop the LIPS assay to capture poultry antibodies generated against PDCoV antigens. Selection and production of capture antigens. To create the PDCoV capture antigens synthetic double stranded DNA oligonucleotides (Integrated DNA Technologies) will be designed against the S1 and N domains of the spike protein from the OH-FD22 strain of PDCoV. Initial alignments utilizing basic local alignment search tool (BLAST) with PDCoV spike amino acid sequence as the query yields 48 coronavirus (CoV) specific hits ranging in identity from 71% (Munia CoV HKU13-3514) to 29% identity for BtRf-AlphaCoVNN2012 with an average identity of 39% (Data not shown). Further breaking down S into the S1 and S2 subdomains shows that S1 (31% Identity, 31 hits) is less conserved than S2 (47% Identity, 52 hits) suggesting S1 is a better target for antigen specificity. Further S1 truncations can result in a S1 fragment with 31% average identity with only 21 hits via BLAST. More in depth analysis may result in even more specific antigen targets. A BLAST search with the N protein of PDCoV results in 50 hits with 34% identity, suggesting N may also be a viable antigen candidate with further sequence evaluation. Several truncated forms of S1 and the N protein will be created to assess antigen specificity. The synthetic DNA fragment will be designed with unique restriction sites to allow insertion of the product into the pNL 1.1 CMV nanoluciferase plasmid (Promega). Plasmids containing the S1, S1-truncation, and N protein reading frames will be transfected intoCos7 cells (ATCC) cultured in DMEM-10% fetal bovine serum using Lipofectamine L TX (lnvitrogen). Recombinant protein quality will be analyzed via denaturing cell lysis and separation using SDS-polyacrylamide gel electrophoresis followed by western blotting using a commercial anti-luciferase antibody. For LIPS assay antigen preparation, two days post transfection cells will be lysed using a solution of 50mM Tris (pH7.5), 100mM NaCl, 5mM MgCl2, 1% Triton X-100, 50% glycerol, and protease inhibitors. Cells scraped from the plate will be sonicated, lysate cleared via 2 centrifugation steps at 12,400RPM. Light units within the cleared lysate will be calculated by adding 1µ1 of lysate to 9µ1 of PBS then adding this mixture directly to 100µ1 of nanoluciferase substrate. The mixture luminescence will be measured immediately using a Filtermax FS plate reader. Aliquots will be stored at -80°C until needed.Experimental serum samples for testing antigen specificity. For positive control samples we will experimentally infect poultry with PDCoV OH-FD22 and allow natural recovery from the virus to generate positive control serum. For specificity control serum poultry will be exposed to the common avian coronavirus infectious bronchitis virus (BEi NR-43284), human CoV NL63 (BEi NR-470), or other available avian coronaviruses such as munia CoV, sparrow CoV, or wigeon CoV. Additionally, serum samples from swine and avian sources that are known to have been infected with specific alpha, beta, gamma, and delta coronaviruses (These archived samples currently exist in the freezers of Linda Saif .) will be evaluated using the LIPS assayto determine if these known circulating CoVs cause cross reactive antibodies to our PDCoV antigens. For experimental infection studies using chickens, turkeys, and ducks. Ten-3 week old chickens, and turkey poults will be obtained from the specific pathogen free flocks maintained on the OARDC campus. Ten eggs of either Muscovy or Pekin breeds will be obtained commercially and hatched at the OARDC facilities. Birds will be divided and housed 5 per room. One room will be inoculated intrachoanally with at least 104 infectious particles. Birds will be monitored daily and allowed to naturally recover from infection. Three to 4 weeks post infection, animals will be exsanguinated and serum will be collected aliquoted and frozen at -80°C until tested. Experimental testing of serum samples with LIPS. Test serum samples will be diluted 1 :100 in buffer A (50 mM Tris, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1% Triton X-100) in a shallow well microtiter plate. Serum samples will be added to a second plate in which a master mix containing the NLuc-antigen extract in which 1 x 107 light units is added to 50µ1 of buffer A. Plates are mixed and incubated for 1 hour at room temperature. The reaction mixture is transferred to a 96 well filter HTS plate (EMD Millipore) containing protein A/G beads for mammalian samples or chicken lgY precipitating resin for avian serum samples. Plates are mixed endover-end for 1 hour at room temperature, washed 8 times with buffer A and 2 times with PBS. Nano Juciferase substrate is added to the wells of the plate, plates are briefly shaken and luminescence is read at 488nm using a Filtermax F5 spectrometer (Molecular Devices). A cutoff value will be determined by comparing luminescence values from test serum of uninfected birds with convalescent PDCoV infected birds. Serum from birds infected with CoVs other than PDCoV will be evaluated to ensure no cross reactivity. Should cross reactivity occur we will attempt to optimize the capture antigen design to reduce cross reactivity. UPS requires minimal assay optimization and, due to its simplicity, quality data can typically be generated using LIPS in under two weeks for any given antigen. The most time consuming steps will be cloning and generating the appropriate plasmid expression vector containing the Niue-antigen fusion and confirming antigen specificity. Once these plasmids are generated, single or multiple antigens can be rapidly tested with LIPS as described above.Aim 2: Evaluate the exposure levels of commercial poultry flocks to PDCoV infection utilizing LIPS. Serum samples for PC'CoV screening. Several collections of serum samples fo1 routine avian influenza virus screening currently exist at the OARDC and Columbus campuses. We will request permission to screen these collections first. If we cannot gain permission to access these collections we will arrange to collect samples from industrial poultry flocks in Ohio. To obtain the field samples 2ml of blood will be taken via the brachia! wing vein using a 20-22 gauge needle on a 5ml syringe and transferred to a 10ml red-top vacutainer tube. Tubes will be allowed to clot at room temperature and stored on ice until processed. For processing, tubes will be spun at 1000RPM for 1 O minutes. Serum will be transferred to 1.5ml tubes and stored at -BOC until processed further. Serum samples will initially be used for LIPS serology. Those serum samples testing positive for seroprevalence to PDCoV antibodies will have RNA extracted from remaining serum using trizol reagent and be subjected to virus screening via RT-PCR using primers specific for the PDCoV genome